Telomeric closure of physical maps of human chromosomes is essential for a comprehensive human genome mapping and sequencing effort. Physically- mapped molecular markers for individual human chromosome termini exist for only a few telomeres, and the distances between the current most distally-mapped markers and chromosome termini remain largely unknown for most human chromosomes. Disease genes which may localize to these regions have few physical or genetic markers for their detection and analysis. Physical mapping of human telomeric DNA will address this gap by characterizing a potentially comprehensive set of large, telomeric yeast artificial-chromosome clones, mapping their distribution in human telomere regions, testing their presumed physical linkage with individual chromosome termini, and developing Sequence-Tagged-Site (STS)-specific Polymerase Chain Reaction (PCR) assays from amongst their sequences. A five-hit coverage human telomeric yeast artificial chromosome (HTY) library of human terminal repeat sequence (T2AG3)n-hybridizing clones (50 kb to 450 kb in size) will be used as source material to develop DNA probes spanning the terminal regions of human chromosomes. These probes, generated from the HTY clones using primarily PCR-based and plasmid-rescue methods, will be used to characterize overlapping HTY clones and assign each group of overlapping clones to an individual telomere. Pulsed-field gel electrophoresis (PFGE) mapping of these probes will test whether the DNA represented in the HTY clones is co- linear with human genomic DNA and physically linked to genomic chromosome termini. These experiments will result in long-range restriction maps for the telomeric regions of most human chromosomes and will reveal general features of human telomeric DNA sequence organization. Relatively short (200 bp to 500 bp) tracts of DNA sequence will be obtained from telomeric DNA probes and used to develop PCR assays. Assays derived from single-copy probes will define STSs; those for low-copy probes will be used to analyze the low-copy sequence family and, if feasible, develop single-copy probes and STS-specific PCR assays for individual members of the low-copy sequence family. The approximately 12 to 16 Mb of cloned telomeric DNA represented in the human telomeric YAC library will span the terminal 250 to 350 kb of most human telomeres, and the proposed experiments will provide both a physical framework for linkage markers derived from these clones and telomeric closure for STS maps of human chromosomes.